Mono- and diacylglycerol acyltransferases and methods of use thereof

ABSTRACT

Nucleic acid compositions encoding polypeptide products with diglyceride acyltransferase and/or monoacylglycerol acyltransferase activity, as well as the polypeptide products encoded thereby, i.e., mammalian DGAT2α and MGAT1 polypeptide products, and methods for producing the same, are provided. Also provided are: methods and compositions for modulating DGAT2α and MGAT1 activity; DGAT2α and MGAT1 transgenic cells, animals and plants, as well as methods for their preparation; and methods for making diglyceride, diglyceride compositions, triglycerides and triglyceride compositions, as well as the compositions produced by these methods. The subject methods and compositions find use in a variety of different applications, including research, medicine, agriculture and industry applications.

CROSS REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/794,715, filed Feb. 26, 2001, which application claimspriority to the filing date of the U.S. Provisional Patent ApplicationSer. No. 60/271,307, filed Feb. 23, 2001, the disclosures of which areherein incorporated by reference in their entirety.

GOVERNMENT RIGHTS

The United States Government may have certain rights in this applicationpursuant to Grant No. DK56084 from the National Institutes of Health.

FIELD OF THE INVENTION

The field of the invention is enzymes, particularly acyltransferases.

BACKGROUND OF THE INVENTION

Diacylglycerol O-Acyltransferase (EC 2.3.1.20), also known asdiglyceride acyltransferase or DGAT, is a critical enzyme intriacylglycerol synthesis. Triacylglycerols are quantitatively the mostimportant storage form of energy for eukaryotic cells. DGAT catalyzesthe rate-limiting and terminal step in triacylglycerol synthesis usingdiacylglycerol and fatty acyl CoA as substrates. As such, DGAT plays afundamental role in the metabolism of cellular diacylglycerol and isimportant in higher eukaryotes for intestinal fat absorption,lipoprotein assembly, fat storage in adipocytes, milk production andpossibly egg production and sperm maturation.

Diacylglycerol is the precursor of such important lipids astriacylglycerol and phospholipids, which store energy and form cellularmembranes. In eukaryotes, two major pathways for synthesizingdiacylglycerol exist: the glycerol phosphate pathway and themonoacylglycerol pathway. Both pathways generate diacylglycerol that canbe used as a substrate by acyl CoA:diacylglycerol acyltransferase (DGAT)for triacylglycerol synthesis. In the glycerol phosphate pathway, whichfunctions in most cells, diacylglycerol is derived by thedephosphorylation of phosphatidic acid produced by sequential acylationsof glycerol phosphate. In the monoacylglycerol pathway, which has beenreported predominantly in the intestine, diacylglycerol is formeddirectly from monoacylglycerol and fatty acyl CoA in a reactioncatalyzed by monoacylglycerol acyltransferase (MGAT) (E.C. 2.3.1.22).

MGAT is best known for its role in fat absorption in the intestine,where the fatty acids and sn-2-monoacylglycerol generated from thedigestion of dietary fat (mainly triacylglycerol) are resynthesized intotriacylglycerol in enterocytes for chylomicron synthesis and secretion.MGAT catalyzes the first step of this process, in which fatty acyl CoA,formed from fatty acids and CoA, and sn-2-monoacylglycerol arecovalently joined. Because the monoacylglycerol pathway predominates inintestinal triacylglycerol synthesis, MGAT may be a pharmaceuticaltarget for modulating fat absorption.

MGAT activity is also found at high levels in liver of suckling rats andin white adipose tissue of migrating sparrows, where triacylglycerolsare actively hydrolyzed to provide fatty acids for energy. MGATpreferentially acylates monoacylglycerols that contain a polyunsaturatedfatty acyl moeity at the sn-2 position. Thus, MGAT may preserveessential fatty acids, all of which are polyunsaturated, byresynthesizing them into triacylglycerols. This function may be relevantin mammalian white adipose tissue, which possesses significant levels ofMGAT activity. In addition, MGAT may also play a role in signaling,since its product, diacylglycerol, and one of its substrates,2-arachidonoylglycerol, are signaling molecules.

Like many enzymes that participate in neutral lipid synthesis, MGAT hasproven difficult to purify to homogeneity, and an MGAT gene has not beenidentified. Several partial purifications of MGAT enzymes have beenreported, and a 43-kDa MGAT enzyme was purified recently from peanutcotyledons. Difficulties in the purification of MGAT may reflect itshydrophobicity or its involvement in an enzyme complex.

Because of its central role in a variety of different processes, thereis much interest in the identification of polynucleotides encodingproteins having DGAT and MGAT activity, as well as the proteins encodedthereby.

Relevant Literature

Of particular interest are: U.S. Pat. No. 6,100,077; and PCT PublishedApplication Nos. WO 98/55631; WO 99/67268; WO 00/01713; WO 99/67403; WO00/32793; WO 00/32756; WO 00/36114; WO 00/60095; WO 00/66749.

Also of interest are: Smith et al., Nat.Genet. 2000 (25), 87–90). Caseset al., “Identification of a gene encoding an acyl CoA:diacylglycerolacyltransferase, a key enzyme in triacylglycerol synthesis,” Proc. Natl.Acad. Sci. USA (October 1998) 95:13018–13023; Oelkers et al.,“Characterization of Two Human Genes Encoding Acyl Coenzyme A:Cholesterol Acyltransferase-Related Enzymes,” J. Biol. Chem. (Oct. 9,1998) 273:26765–71; and Cases et al. (2001) J. Biol. Chem.276:38870–38876.

References describing the role DGAT plays in various biologicalprocesses include: Bell & Coleman, “Enzymes of Glycerolipid Synthesis inEukaryotes,” Annu. Rev. Biochem. (1980) 49: 459–487; Lehner & Kuksis,“Biosynthesis of Triacylglycerols,” Prog. Lipid Res. (1996) 35: 169–201;Brindley, Biochemistry of Lipids, Lipoproteins and Membranes (eds. Vance& Vance) (Elsevier, Amsterdam) (1991) pp 171–203; Haagsman & Van Golde,“Synthesis and Secretion of Very Low Density Lipoproteins by IsolatedRat Hepatocytes in Suspension: Role of Diacylglycerol Acyltransferase,”Arch. Biochem. Biophys. (1981) 208:395–402; Coleman & Bell,“Triacylglycerol Synthesis in Isolated Fat Cells. Studies on theMicrosomal Diacylglycerol Acyltransferase Activity UsingEthanol-Dispersed Diacylglycerols,” J. Biol. Chem. (1976) 251:4537–4543.

References discussing MGAT activity and purification include: Colemanand Haynes (1984) J. Biol. Chem. 259:8934–8938; Mostafa et al. (1994)Lipids 29:785–791; Xia et al. 1993) Am. J. Physiol. 265:R414–R419;Jamdar et al. (1992) Arch. Biochem. Biophys. 296:419–425; Manganaro etal. (1985) Can. J. Biochem. Cell Biol. 63:341–347; Bhat et al. (1993)Arch. Biochem. Biophys. 300:663–669; Tumaney et al. (2001) J. Biol.Chem. 276:10847–10852; Lehner and Kuksis (1995) J. Biol. Chem.270:13630–13636.

SUMMARY OF THE INVENTION

Nucleic acid compositions encoding polypeptide products with diglycerideacyltransferase and/or monoacylglycerol acyltransferase activity, aswell as the polypeptide products encoded thereby, i.e., mammalian DGAT2αand MGAT1 polypeptide products, and methods for producing the same, areprovided. Also provided are: methods and compositions for modulatingDGAT2α and MGAT1 activity; DGAT2α and MGAT1 transgenic cells, animalsand plants, as well as methods for their preparation; and methods formaking diglyceride, diglyceride compositions, triglycerides andtriglyceride compositions, as well as the compositions produced by thesemethods. The subject methods and compositions find use in a variety ofdifferent applications, including research, medicine, agriculture andindustry applications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a graphical representation of the results obtained froma pulse assay that demonstrates the existence of mouse DGAT2α.

FIG. 2 provides a hydrophobicity plot of mouse DGAT2α.

FIGS. 3A to 3C provide graphical results of various mouse DGAT2αactivity assays.

FIG. 4 provides the expression profile for mouse DGAT2α.

FIG. 5 provides the results of an assay showing that mouse DGAT2αexpression increases during 3T3-L1 adipocyte differentiation.

FIGS. 6A and 6B provide the amino acid and nucleic acid sequences ofmouse DGAT2α.

FIGS. 7A and 7B provide the amino acid and nucleic acid sequences ofhuman DGAT2α.

FIGS. 8A–D provide the amino acid and nucleic acid sequences of variousmouse and human DGAT2α homologs, including a mouse DC2 amino acidsequence (SEQ ID NO:06), a mouse DC2 nucleic acid sequence (SEQ IDNO:5), a human DC2 amino acid sequence (SEQ ID NO:08), a human DC2nucleic acid sequence (SEQ ID NO:07), a mouse DC3 amino acid sequence(SEQ ID NO:10), a mouse DC3 nucleic acid sequence (SEQ ID NO:09), ahuman DC3 amino acid sequence (SEQ ID NO:12), a human nucleic acidsequence (SEQ ID NO:11), a human DC4 amino acid sequence (SEQ ID NO:14),a human DC4 nucleic acid sequence (SEQ ID NO:13), a human DC5 amino acidsequence (SEQ ID NO:16), and a human DC5 nucleic acid sequence (SEQ IDNO:15).

FIG. 9 depicts schematically two major pathways for synthesizingdiacylglycerol (DAG).

FIG. 10A depicts a comparison of the amino acid sequences of mouse MGAT1(SEQ ID NO:06) and mouse DGAT2 (SEQ ID NO:04). FIG. 10B depicts ahydrophobicity plot of mouse MGAT1.

FIG. 11 depicts graphs showing MGAT1 activity using oleoyl CoA or2-monooleoylglycerol.

FIG. 12 depicts graphs showing that MGAT1 has activity toward allstereoisomers of monoacyl glycerol.

FIG. 13 depicts expression of MGAT1 protein in COS-7 cells.

FIG. 14 depicts the tissue distribution of MGAT1 mRNA expression.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic acid compositions encoding polypeptide products with diglycerideand/or monoacylglycerol acyltransferase activity, as well as thepolypeptide products encoded thereby, i.e., mammalian DGAT2α and MGAT1,and methods for producing the same, are provided. In many embodiments,the subject nucleic acids encode enzymes that exhibit monoacylglycerolacyltransferase activity, diacylglycerol acyltransferase activity, orboth mono- and diacyltransferase activity. For example, DGAT2αpolypeptides exhibit diglyceride acyltransferase activity (also referredto herein as “DGAT2” polypeptides); and mammalian MGAT1 polypeptides(also referred to herein as “DC2” polypeptides) exhibit monoacylglycerolacyltransferase activity, and in some embodiments also exhibitdiacylglycerol acyltransferase activity.

Also provided are: methods and compositions for modulating DGAT2α andMGAT1 activity, e.g. in the treatment of disease conditions associatedwith DGAT2α and/or MGAT1 activity, including obesity; MGAT1 and DGAT2αtransgenic cells, animals, plants and fungi, and methods for theirpreparation, e.g. for use in research, food production, industrialfeedstock production, etc.; and methods for making diglycerides,diglyceride compositions, triglycerides, and triglyceride compositions,e.g. oils. The methods and compositions of the subject invention finduse in a variety of different applications and fields, includingresearch, medicine, agriculture and industry.

Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Nucleic Acid Compositions

Nucleic acid compositions encoding polypeptide products, as well asfragments thereof, having mono- and/or diglyceride acetyltransferaseactivity are provided. In many embodiments, the subject nucleic acidsencode enzymes that exhibit monoacylglycerol acyltransferase activity,diacylglycerol acyltransferase activity, or both mono- anddiacyltransferase activity. Specifically, nucleic acid compositionsencoding mammalian, e.g., human, mouse, etc., DGAT2α polypeptides havingdiglyceride acyltransferase activity (also referred to herein as “DGAT2”polypeptides), and mammalian MGAT1 polypeptides exhibitingmonoacylglycerol acyltransferase activity (also referred to herein as“DC2” polypeptides), are provided.

By nucleic acid composition is meant a composition comprising a sequenceof DNA having an open reading frame that encodes a DGAT2α or an MGAT1polypeptide, i.e. a gene or genomic region encoding a polypeptide havingmono- and/or diglyceride acyltransferase activity, and is capable, underappropriate conditions, of being expressed as a DGAT2α or an MGAT1polypeptide.

Also encompassed in this term are nucleic acids that are homologous orsubstantially similar or identical to the nucleic acids encoding DGAT2αpolypeptides, or MGAT1 polypeptides. Thus, the subject inventionprovides nucleic acids encoding mammalian DGAT2α, such as nucleic acidsencoding human DGAT2α and homologs thereof and mouse DGAT2α and homologsthereof. The subject invention provides nucleic acids encoding mammalianMGAT1, such as nucleic acids encoding mouse MGAT1 (also referred toherein as “DC2”), and homologs thereof.

The coding sequence of the human DGAT2α genomic sequence, i.e. the humancDNA encoding the human DGAT2α enzyme, includes or comprises a nucleicacid sequence substantially the same as or identical to that identifiedas SEQ ID NO:01 or SEQ ID NO:18, infra. The coding sequence of the mouseDGAT2α genomic sequence, i.e., the mouse cDNA encoding the mouse DGAT2αenzyme, includes or comprises a nucleic acid substantially the same asor identical to the sequence identified as SEQ ID NO:03, infra. Thecoding sequence of the mouse MGAT1 genomic sequence, i.e. the mouse cDNAencoding the mouse MGAT1 enzyme, includes or comprises a nucleic acidsequence substantially the same as or identical to that identified asSEQ ID NO:05, infra.

The source of homologous nucleic acids to those specifically listedabove may be any species, including both animal and plant species, e.g.,primate species, particularly human; rodents, such as rats and mice,canines, felines, bovines, ovines, equines, yeast, nematodes, etc.Between mammalian species, e.g., human and mouse, homologs havesubstantial sequence similarity, e.g. at least 75% sequence identity,usually at least 90%, more usually at least 95% between nucleotidesequences. Sequence similarity is calculated based on a referencesequence, which may be a subset of a larger sequence, such as aconserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990), J. Mol. Biol.215:403–10. Unless specified otherwise, all sequence identity valuesprovided herein are determined using GCG (Genetics Computer Group,Wisconsin Package, Standard Settings, gap creation penalty 3.0, gapextension penalty 0.1). The sequences provided herein are essential forrecognizing DGAT2α-related and homologous polynucleotides in databasesearches. Specific DGAT2α homologues of interest are provide in FIG. 8,i.e., SEQ ID NOs. 05, 07, 09, 11, 13 and 15.

Also provided are nucleic acids that hybridize to the above-describedspecific nucleic acids, e.g., those nucleic acids having a sequence ofSEQ ID NO:01, 03, 05, 07, 09, 11, 13, 15, or 18, or a coding sequence ofany one of the foregoing sequences, under stringent conditions. Anexample of stringent hybridization conditions is hybridization at 50° C.or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate).Another example of stringent hybridization conditions is overnightincubation at 42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl,15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.Stringent hybridization conditions are hybridization conditions that areat least as stringent as the above representative conditions. Otherstringent hybridization conditions are known in the art and may also beemployed to identify nucleic acids of this particular embodiment of theinvention.

Also provided are nucleic acids that encode a polypeptide having monoand/or diacylglycerol acyltransferase activity and having at least about50%, at least about 60%, at least about 70&, at least about 75%, atleast about 80%, at least about 90%, at least about 95%, or higher,nucleotide sequence identity to a nucleic acid having a nucleic acidsequence set forth in any one of SEQ ID NO:01, 03, 05, 07, 09, 11, 13,15, or 18. Also provided are nucleic acids that encode a polypeptidehaving mono- and/or diacylglycerol acyltransferase activity and havingat least about 50%, at least about 60%, at least about 70&, at leastabout 75%, at least about 80%, at least about 90%, at least about 95%,or higher, nucleotide sequence identity to the coding region of anucleic acid having a sequence set forth in any one of SEQ ID NO:01, 03,05, 07, 09, 11, 13, 15, or 18.

Nucleic acids encoding the DGAT2α proteins, DGAT2α polypeptides, andMGAT polypeptides of the subject invention may be cDNAs or genomic DNAs,i.e. portions of chromosomes that include both introns and exons, aswell as promoter regions, etc., as well as fragments thereof. The term“DGAT2α-gene” shall be intended to mean the open reading frame encodingspecific DGAT2α proteins and polypeptides, and DGAT2α introns, as wellas adjacent 5′ and 3′ non-coding nucleotide sequences involved in theregulation of expression, up to about 20 kb beyond the coding region,but possibly further in either direction. Similarly, the term “MGAT1gene” refers to the open reading from encoding specific MGATpolypeptides, and MGAT1 introns, as well as adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression, up to about 20 kb beyond the coding region, but possiblyfurther in either direction. The gene may be introduced into anappropriate vector for extrachromosomal maintenance or for integrationinto a host genome.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 3′ and 5′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding an MGAT1 ora DGAT2α protein.

Also provided are nucleic acids that encode the DGAT2α and MGAT1proteins encoded by the above described nucleic acids, but differ insequence from the above described nucleic acids due to the degeneracy ofthe genetic code. Also provided are nucleic acids that encode DGAT2α andMGAT1 proteins that include conservative amino acid changes whencompared to, e.g., the amino acid sequences set forth in SEQ ID NO:02 or06.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3′ and 5′untranslated regions found in the mature mRNA. It may further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

The nucleic acid compositions of the subject invention may encode all ora part of the subject DGAT2α or MGAT1 proteins and polypeptides,described in greater detail infra. Double or single stranded fragmentsmay be obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least 15 nt, usually at least 18 nt or 25 nt,and may be at least about 50 nt.

The MGAT1 and DGAT2α-nucleic acids or genes of the subject invention areisolated and obtained in substantial purity, generally as other than anintact chromosome. Usually, the DNA will be obtained substantially freeof other nucleic acid sequences that do not include a DGAT2α sequence orfragment thereof, generally being at least about 50%, usually at leastabout 90% pure and are typically “recombinant”, i.e. flanked by one ormore nucleotides with which it is not normally associated on a naturallyoccurring chromosome.

In addition to the plurality of uses described in greater detail infollowing sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the DGAT2α or MGAT1 polypeptides,as described below.

Polypeptide Compositions

Also provided by the subject invention are polypeptides having mono-and/or diglyceride acyltransferase activity, i.e., capable of catalyzingthe acylation of diacylglycerol, acylation of monoacylglycerol, oracylation of both mono- and diacylglycerol. Such enzymes are referred toherein as “mono- and diacylglcerol acyltransferases.” Examples of suchpolypeptides are DGAT2α (also referred to as “DGAT2”) and MGAT1 (alsoreferred to as “DC2”). The term “polypeptide composition” as used hereinrefers to both full-length proteins as well as portions or fragmentsthereof. Also included in this term are variations of the naturallyoccurring proteins, where such variations are homologous orsubstantially similar to the naturally occurring protein, as describedin greater detail below, be the naturally occurring protein the humanprotein, mouse protein, or protein from some other mammalian specieswhich naturally expresses a subject acyltransferase. In the followingdescription of the subject invention, the term “DGAT2α” is used to refernot only to the human form of the enzyme, but also to homologs thereofexpressed in non-human mammalian species. Similarly, the term “mouseMGAT1” refers not only to the mouse form of the enzyme, but also tohomologs thereof expressed in other mammalian species.

The subject mono- and diacylglcerol acyltransferases are, in theirnatural environment, trans-membrane proteins. The subject proteins arecharacterized by the presence of at least one potential N-linkedglycosylation site, at least one potential tyrosine phosphorylationsite, and multiple hydrophobic domains, including 4 to 12, e.g., 6,hydrophobic domains capable of serving as trans-membrane regions. Theproteins range in length from about 300 to 500, usually from about 325to 475 and more usually from about 350 to 425 amino acid residues, andthe projected molecular weight of the subject proteins based solely onthe number of amino acid residues in the protein ranges from about 35 to55, usually from about 37.5 to 47.5 and more usually from about 40 to 45kDa, where the actual molecular weight may vary depending on the amountof glycolsylation of the protein and the apparent molecular weight maybe considerably less because of SDS binding on gels.

The amino acid sequences of the subject proteins are characterized byhaving substantially no homology to the known DGAT enzymes. Morespecifically, the subject human DGAT2α and mouse MGAT1 enzymes havesubstantially no homology to the human DGAT enzyme described in Cases etal., “Identification of a gene encoding an acyl CoA:diacylglycerolacyltransferase, a key enzyme in triacylglycerol synthesis,” Proc. Natl.Acad. Sci. U.S.A. 95 (22), 13018–13023 (1998). Likewise, the subjectmouse DGAT2α enzymes have substantially no homology to the mouse DGATenzyme described in Cases et al., “Identification of a gene encoding anacyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerolsynthesis,” Proc. Natl. Acad. Sci. U.S.A. 95 (22), 13018–13023 (1998).By substantially no homology is meant that the homology does not exceedabout 20%, and usually will not exceed about 10% and more usually willnot exceed about as determined using GCG (Genetics Computer Group,Wisconsin Package, Standard Settings, Gap Creation Penalty 3.0, GapExtension Penalty 0.1).

Of particular interest in many embodiments are proteins that arenon-naturally glycosylated. By non-naturally glycosylated is meant thatthe protein has a glycosylation pattern, if present, which is not thesame as the glycosylation pattern found in the corresponding naturallyoccurring protein. For example, human DGAT2α of the subject inventionand of this particular embodiment is characterized by having aglycosylation pattern, if it is glycosylated at all, that differs fromthat of naturally occurring human DGAT2α. Thus, the non-naturallyglycosylated DGAT2α proteins of this embodiment include non-glycosylatedDGAT2α proteins, i.e. proteins having no covalently bound glycosylgroups.

The sequence of the full-length human DGAT2α protein is identified,infra, as SEQ ID NO:02. As such, DGAT2α proteins having an amino acidsequence that is substantially the same as or identical to the sequenceof SEQ ID NO:2 are of interest. By substantially the same as is meant aprotein having a region with a sequence that has at least about 75%,usually at least about 90% and more usually at least about 98% sequenceidentity with the sequence of SED ID NO:02, as measured by GCG, supra.Of particular interest in other embodiments is the mouse DGAT2α protein,where the mouse DGAT2α protein of the subject invention has an aminoacid sequence that is substantially the same as or identical to thesequence appearing as SEQ ID NO:04, infra.

In addition to the specific mammalian DGAT2α proteins described above,homologs or proteins (or fragments thereof) from other species, i.e.other animal or plant species, are also provided, where such homologs orproteins may be from a variety of different types of species, includinganimals, such as mammals, e.g., rodents, such as rats, mice; domesticanimals, e.g. horse, cow, dog, cat; humans, and the like. By homolog ismeant a protein having at least about 35%, usually at least about 40%and more usually at least about 60% amino acid sequence identity thespecific DGAT2α proteins as identified in SEQ ID NOS: 02 to 04, wheresequence identity is determined using GCG, supra. Specific homologs ofinterest include human DC2, human DC3, human DC4, human DC5, mouse DC2and mouse DC3, the sequences of which are provided in FIG. 8 (i.e., SEQID NOs. 06, 08, 10, 12, 14 and 16).

Mouse MGAT1 exhibits monoacylglycerol acyltransferase activity. Thesequence of the full-length mouse MGAT1 protein is identified, infra, asSEQ ID NO:06 (identified as “mouse DC2” in FIG. 8). As such, subjectMGAT1 proteins having an amino acid sequence that is substantially thesame as or identical to the sequence of SEQ ID NO:06 are of interest. Bysubstantially the same as is meant a protein having a region with asequence that has at least about 75%, usually at least about 90% andmore usually at least about 98% sequence identity with the sequence ofSED ID NO:06, as measured by GCG, supra.

Mono- and diacylglcerol acyltransferases of the subject invention (e.g.human DGAT2α or a homolog thereof; non-human DGAT2α proteins, e.g. mouseDGAT2α; mouse MGAT1 polypeptide or a homolog thereof) are present in anon-naturally occurring environment, e.g. are separated from theirnaturally occurring environment. In certain embodiments, the subjectmono- and diacylglcerol acyltransferases are present in a compositionthat is enriched for such an enzyme, e.g., enriched for DGAT2α ascompared to DGAT2α in its naturally occurring environment. As such,purified mono- and diacylglcerol acyltransferases are provided, where bypurified is meant that subject enzyme is present in a composition thatis substantially free of proteins other than the subject enzyme, whereby substantially free is meant that less than 90%, usually less than 60%and more usually less than 50% of the composition is made up of proteinsother than the subject enzyme. For example, for compositions that areenriched for DGAT2α proteins, such compositions will exhibit a DGAT2αactivity of at least about 100, usually at least about 200 and moreusually at least about 1000 pmol triglycerides formed/mg protein/min,where such activity is determined by the assay described in theExperimental Section, infra.

In certain embodiments of interest, a subject enzyme is present in acomposition that is substantially free of the constituents that arepresent in its naturally occurring environment. For example, a humanDGAT2α protein comprising composition according to the subject inventionin this embodiment will be substantially, if not completely, free ofthose other biological constituents, such as proteins, carbohydrates,lipids, etc., with which it is present in its natural environment. Assuch, protein compositions of these embodiments will necessarily differfrom those that are prepared by purifying the protein from a naturallyoccurring source, where at least trace amounts of the protein'sconstituents will still be present in the composition prepared from thenaturally occurring source.

The mono- and diacylglcerol acyltransferases of the subject inventionmay also be present as an isolate, by which is meant that the subjectenzyme is substantially free of both proteins other than a subjectenzyme and other naturally occurring biologic molecules, such asoligosaccharides, polynucleotides and fragments thereof, and the like,where substantially free in this instance means that less than 70%,usually less than 60% and more usually less than 50% (dry weight) of thecomposition containing the isolated subject enzyme is a naturallyoccurring biological molecule other than the subject enzyme. In certainembodiments, the subject enzyme is present in substantially pure form,where by substantially pure form is meant at least 95%, usually at least97% and more usually at least 99% pure.

In addition to the naturally occurring subject proteins, mono- anddiacylglcerol acyltransferase polypeptides which vary from the naturallyoccurring DGAT2α and/or MGAT1 proteins are also provided. By “DGAT2αpolypeptides” and “MGAT1 polypeptides” is meant proteins having an aminoacid sequence encoded by an open reading frame (ORF) of a DGAT2α gene oran MGAT1 gene, respectively, as described supra, including the fulllength DGAT2α or MGAT1 protein and fragments thereof, particularlybiologically active fragments and/or fragments corresponding tofunctional domains; and including fusions of the subject polypeptides toother proteins or parts thereof. Fragments of interest will typically beat least about 10 amino acids (aa) in length, usually at least about 50aa in length, and may be as long as 300 aa in length or longer, but willusually not exceed about 1000 aa in length, where the fragment will havea stretch of amino acids that is identical to a subject protein of SEQID NO:2, SEQ ID NO:04, or SEQ ID NO:06 or a homolog thereof; of at leastabout 10 aa, and usually at least about 15 aa, and in many embodimentsat least about 50 aa in length.

Preparation of Subject Polypeptides

The subject proteins and polypeptides may be obtained from naturallyoccurring sources, but are preferably synthetically produced. Whereobtained from naturally occurring sources, the source chosen willgenerally depend on the species from which the subject protein is to bederived.

The subject polypeptide compositions may be synthetically derived byexpressing a recombinant gene encoding the subject protein, such as thepolynucleotide compositions described above, in a suitable host. Forexpression, an expression cassette may be employed. The expressionvector will provide a transcriptional and translational initiationregion, which may be inducible or constitutive, where the coding regionis operably linked under the transcriptional control of thetranscriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native toa DGAT2α gene, an MGAT1 gene, or may be derived from exogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fusionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzymemarker, e.g. β-galactosidase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading-frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

Subject proteins and polypeptides may be expressed in prokaryotes oreukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, may be used as the expression host cells. In some situations, itis desirable to express the subject coding sequence in eukaryotic cells,where the DGAT2α or MGAT1 protein will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory. Polypeptides that are subsets of the complete DGAT2αor MGAT1 sequence may be used to identify and investigate parts of theprotein important for function.

Once the source of the protein is identified and/or prepared, e.g. atransfected host expressing the protein is prepared, the protein is thenpurified to produce the desired DGAT2α- or MGAT1-comprising composition.Any convenient protein purification procedures may be employed, wheresuitable protein purification methodologies are described in Guide toProtein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may prepared from the original source, e.g. naturallyoccurring cells or tissues that express DGAT2α or the expression hostexpressing DGAT2α, and purified using HPLC, exclusion chromatography,gel electrophoresis, affinity chromatography, and the like.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories is are provided below:

Bacteria. Expression systems in bacteria include those described inChang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21–25; and Siebenlist et al., Cell (1980) 20:269.

Yeast. Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284–289;Tilburn et al., Gene (1983) 26:205–221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470–1474; Kelly and Hynes, EMBO J. (1985)4:475479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051; Friesen et al.,“The Regulation of Baculovirus Gene Expression”, in: The MolecularBiology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765–776; Miller etal., Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988)73:409; Maeda et al., Nature (1985) 315:592–594; Lebacq-Verheyden etal., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad.Sci. (USA) (1985) 82:8844; Miyajima et al., Gene (1987) 58:273; andMartin et al., DNA (1988) 7:99. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts aredescribed in Luckow et al., Bio/Technology (1988) 6:47–55, Miller etal., Generic Engineering (1986) 8:277–279, and Maeda et al., Nature(1985) 315:592–594.

Mammalian Cells. Mammalian expression is accomplished as described inDijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad.Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S.Pat. No. 4,399,216. Other features of mammalian expression arefacilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195,and U.S. RE No. 30,985.

When any of the above host cells, or other appropriate host cells ororganisms, are used to replicate and/or express the polynucleotides ornucleic acids of the invention, the resulting replicated nucleic acid,RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism. The product isrecovered by any appropriate means known in the art.

Once the gene corresponding to a selected polynucleotide is identified,its expression can be regulated in the cell to which the gene is native.For example, an endogenous gene of a cell can be regulated by anexogenous regulatory sequence as disclosed in U.S. Pat. No. 5,641,670.

Methods and Compositions Having Research Application

Also provided by the subject invention are methods and compositionshaving research applications, such as in the study of the acylglycerolmetabolism; in the identification of key components of the di- andtriglyceride synthesis pathway; in the identification of di- andtriglyceride synthesis modulatory agents, e.g. DGAT2α or MGAT1inhibitors or enhancers, and the like.

The subject nucleic acid compositions find use in a variety of researchapplications. Research applications of interest include: theidentification of DGAT2α and MGAT1 homologs; as a source of novelpromoter elements; the identification of DGAT2α or MGAT1 expressionregulatory factors; as probes and primers in hybridization applications,e.g. PCR; the identification of expression patterns in biologicalspecimens; the preparation of cell or animal models for DGAT2α or MGAT1function; the preparation of in vitro models for DGAT2α or MGAT1function; etc.

Homologs of the specifically disclosed subject nucleic acids areidentified by any of a number of methods. A fragment of the providedcDNA may be used as a hybridization probe against a cDNA library fromthe target organism of interest, where low stringency conditions areused. The probe may be a large fragment, or one or more short degenerateprimers. Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 6×SSC (0.9 M sodium chloride/0.09 M sodium citrate) and remain boundwhen subjected to washing at 55° C. in 1×SSC (0.15 M sodiumchloride/0.015 M sodium citrate). Sequence identity may be determined byhybridization under stringent conditions, for example, at 50° C. orhigher and 0.1×SSC (15 mM sodium chloride/01.5 mM sodium citrate).Nucleic acids having a region of substantial identity to the providednucleic acid sequences bind to the provided sequences under stringenthybridization conditions. By using probes, particularly labeled probesof DNA sequences, one can isolate homologous or related genes. One canalso use sequence information derived from the polynucleotidecompositions of the subject invention to prepare electronic “probes” foruse in searching of computer based sequence date, e.g. BLAST searchesEST databases.

The sequence of the 5′ flanking region of the subject nucleic acidcompositions may be utilized as a source for promoter elements,including enhancer-binding sites, that provide for developmentalregulation in tissues where a subject acyltransferase, e.g., DGAT2α orMGAT1, is expressed. The tissue-specific expression is useful fordetermining the pattern of expression, and for providing promoters thatmimic the native pattern of expression. Naturally occurringpolymorphisms in the promoter region are useful for determining naturalvariations in expression, particularly those that may be associated withdisease.

Alternatively, mutations may be introduced into the promoter region todetermine the effect of altering expression in experimentally definedsystems. Methods for the identification of specific DNA motifs involvedin the binding of transcriptional factors are known in the art, e.g.sequence similarity to known binding motifs, gel retardation studies,etc. For examples, see Blackwell et al. (1995), Mol. Med. 1:194–205;Mortlock et al. (1996), Genome Res. 6:327–33; and Joulin and Richard-Foy(1995), Eur. J. Biochem. 232:620–626.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of DGAT2 geneexpression, especially in different tissues or stages of development,and to identify cis acting sequences and trans-acting factors thatregulate or mediate DGAT2 or MGAT1 gene expression. Such transcriptionor translational control regions may be operably linked to a DGAT2 orMGAT gene in order to promote expression of wild type or altered DGAT2or MGAT1 or other proteins of interest in cultured cells, or inembryonic, fetal or adult tissues, and for gene therapy.

Small DNA fragments are useful as primers for PCR, hybridizationscreening probes, etc. Larger DNA fragments, i.e. greater than 100nucleotides (nt) are useful for production of the encoded polypeptide,as described in the previous section. For use in amplificationreactions, such as PCR, a pair of primers will be used. The exactcomposition of the primer sequences is not critical to the invention,but for most applications the primers will hybridize to the subjectsequence under stringent conditions, as known in the art. It ispreferable to choose a pair of primers that will generate anamplification product of at least about 50 nt, preferably at least about100 nt. Algorithms for the selection of primer sequences are generallyknown, and are available in commercial software packages. Amplificationprimers hybridize to complementary strands of DNA, and will primetowards each other.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The mRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of DGAT2α or MGAT1 gene expression in the sample.

The sequence of a subject gene or nucleic acid, including flankingpromoter regions and coding regions, may be mutated in various waysknown in the art to generate targeted changes in promoter strength,sequence of the encoded protein, etc. The DNA sequence or proteinproduct of such a mutation will usually be substantially similar to thesequences provided herein, i.e. will differ by at least one nucleotideor amino acid, respectively, and may differ by at least two but not morethan about ten nucleotides or amino acids. The sequence changes may besubstitutions, insertions, deletions, or a combination thereof.Deletions may further include larger changes, such as deletions of adomain or exon. Other modifications of interest include epitope tagging,e.g. with the FLAG system, HA, etc. For studies of subcellularlocalization, fusion proteins with green fluorescent proteins (GFP) maybe used.

Techniques for in vitro mutagenesis of cloned genes are known. Examplesof protocols for site specific mutagenesis may be found in Gustin et al.(1993), Biotechniques 14:22; Barany (1985), Gene 37:111–23; Colicelli etal. (1985), Mol. Gen. Genet. 199:537–9; and Prentki et al. (1984), Gene29:303–13. Methods for site specific mutagenesis can be found inSambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 15.3–15.108; Weiner et al. (1993), Gene 126:35–41; Sayers et al.(1992), Biotechniques 13:592–6; Jones and Winistorfer (1992),Biotechniques 12:528–30; Barton et al. (1990), Nucleic Acids Res18:7349–55; Marotti and Tomich (1989), Gene Anal. Tech. 6:67–70; and Zhu(1989), Anal Biochem 177:120–4. Such mutated genes may be used to studystructure-function relationships of DGAT2α, or to alter properties ofthe protein that affect its function or regulation.

The subject nucleic acids can be used to generate transgenic hosts, e.g.non-human animals, such as mice, cows, rats, pigs etc., or site specificgene modifications in cell lines. Examples of transgenic hosts includehosts in which the naturally expressed DGAT2α or MGAT1 gene has beendisrupted, e.g. DGAT2α or MGAT1 knock-outs, as well as hosts in whichDGAT2α or MGAT1 expression has been amplified, e.g. through introductionof additional DGAT2α or MGAT1 copies, through introduction of strongpromoter upstream of the DGAT2α or MGAT1 gene, and the like. Using thenucleic acid compositions of the subject invention, standard protocolsknown to those of skill in the art may used to produce such transgenichosts that have been genetically manipulated with respect to the subjectgene, i.e. DGAT2α or MGAT1 transgenic hosts.

Transgenic animals may be made through homologous recombination, wherethe normal DGAT2α or MGAT1 locus is altered, e.g. as in DGAT2α or MGAT1knockouts. Alternatively, a nucleic acid construct is randomlyintegrated into the genome. Vectors for stable integration includeplasmids, retroviruses and other animal viruses, YACs, and the like. DNAconstructs for homologous recombination will comprise at least a portionof the DGAT2α or MGAT1 gene native to the species of the host animal,wherein the gene has the desired genetic modification(s), and includesregions of homology to the target locus. DNA constructs for randomintegration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included. Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.(1990), Meth. Enzymol. 185:527–537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, cow, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The resultant chimeric animals are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogeneic or congenic grafts or transplants, or in in vitro culture.The transgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc.

Transgenic plants may be produced in a similar manner. Methods ofpreparing transgenic plant cells and plants are described in U.S. Pat.Nos. 5,767,367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731;5,656,466; 5,633,155; 5,629,470; 5,595,896; 5,576,198; 5,538,879;5,484,956; the disclosures of which are herein incorporated byreference. Methods of producing transgenic plants are also reviewed inPlant Biochemistry and Molecular Biology (eds Lea & Leegood, John Wiley& Sons)(1993) pp 275–295. In brief, a suitable plant cell or tissue isharvested, depending on the nature of the plant species. As such, incertain instances, protoplasts will be isolated, where such protoplastsmay be isolated from a variety of different plant tissues, e.g. leaf,hypoctyl, root, etc. For protoplast isolation, the harvested cells areincubated in the presence of cellulases in order to remove the cellwall, where the exact incubation conditions vary depending on the typeof plant and/or tissue from which the cell is derived. The resultantprotoplasts are then separated from the resultant cellular debris bysieving and centrifugation. Instead of using protoplasts, embryogenicexplants comprising somatic cells may be used for preparation of thetransgenic host. Following cell or tissue harvesting, exogenous DNA ofinterest is introduced into the plant cells, where a variety ofdifferent techniques are available for such introduction. With isolatedprotoplasts, the opportunity arise for introduction via DNA-mediatedgene transfer protocols, including: incubation of the protoplasts withnaked DNA, e.g. plasmids, comprising the exogenous coding sequence ofinterest in the presence of polyvalent cations, e.g. PEG or PLO; andelectroporation of the protoplasts in the presence of naked DNAcomprising the exogenous sequence of interest. Protoplasts that havesuccessfully taken up the exogenous DNA are then selected, grown into acallus, and ultimately into a transgenic plant through contact with theappropriate amounts and ratios of stimulatory factors, e.g. auxins andcytokinins. With embryogenic explants, a convenient method ofintroducing the exogenous DNA in the target somatic cells is through theuse of particle acceleration or “gene-gun” protocols. The resultantexplants are then allowed to grow into chimera plants, cross-bred andtransgenic progeny are obtained. Instead of the naked DNA approachesdescribed above, another convenient method of producing transgenicplants is Agrobacterium mediated transformation. With Agrobacteriummediated transformation, co-integrative or binary vectors comprising theexogenous DNA are prepared and then introduced into an appropriateAgrobacterium strain, e.g. A. tumefaciens. The resultant bacteria arethen incubated with prepared protoplasts or tissue explants, e.g. leafdisks, and a callus is produced. The callus is then grown underselective conditions, selected and subjected to growth media to induceroot and shoot growth to ultimately produce a transgenic plant.

The modified cells, animals or plants are useful in the study offunction and regulation or a subject gene or nucleic acid. For example,a series of small deletions and/or substitutions may be made in thehost's native DGAT2α or MGAT1 gene to determine the role of differentexons in various physiological processes. Specific constructs ofinterest include anti-sense nucleic acids, which will block DGAT2α orMGAT1 expression, expression of dominant negative DGAT2α or MGAT1mutations, and over-expression of DGAT2α or MGAT1 genes. Where a subjectnucleic acid sequence is introduced, the introduced sequence may beeither a complete or partial sequence of a subject gene native to thehost, or may be a complete or partial subject nucleic acid sequence thatis exogenous to the host animal, e.g., a human DGAT2α or a human MGAT1sequence. A detectable marker, such as lac Z (encoding β-galactosidase)may be introduced into the DGAT2α or MGAT1 locus, where upregulation ofDGAT2α or MGAT1 gene expression will result in an easily detected changein phenotype. One may also provide for expression of the subject gene orvariants thereof in cells or tissues where it is not normally expressed,at levels not normally present in such cells or tissues, or at abnormaltimes of development. The transgenic hosts, e.g. animals, plants, etc.,may be used in functional studies, drug screening, etc., e.g. todetermine the effect of a candidate drug on DGAT2α or MGAT1 activity, toidentify drugs that reduce serum triglyceride levels, etc.

The subject polypeptide compositions can be used to produce in vitromodels of diglyceride and/or triglyceride synthesis, where such modelswill consist of the subject proteins and other components of di- and/ortriglyceride synthesis, e.g. substrates, such as monoacylglycerol,diacylglycerol or metabolic precursors thereof, fatty acyl CoAs and thelike, other components of the diacylglycerol and/or triacylglycerolsynthetase complex, e.g. acyl CoA ligase, acyl CoA acyltransferase,monoacyl glycerol acyltransferase, etc.

Also provided by the subject invention are screening assays designed tofind modulatory agents of activity of a subject mono- or diacylglycerolacyltransferase, e.g. inhibitors or enhancers of DGAT2α or MGAT1activity, as well as the agents identified thereby, where such agentsmay find use in a variety of applications, including as therapeuticagents, as agricultural chemicals, etc. The screening methods willtypically be assays which provide for qualitative/quantitativemeasurements of DGAT2α or MGAT1 activity in the presence of a particularcandidate therapeutic agent. For example, the assay could be an assaywhich measures the acylation activity of DGAT2α or MGAT1 in the presenceand absence of a candidate inhibitor agent. The screening method may bean in vitro or in vivo format, where both formats are readily developedby those of skill in the art.

Thus, in some embodiments, the invention provides an in vitro method ofidentifying an agent that modulates the acyltransferase activity ofMGAT1. The method generally involves contacting MGAT1 with a candidateagent (also referred to as a “test agent”) in the presence of an acyldonor and an acyl acceptor. The effect, if any, of a test agent on theamount of acylated acceptor that is produced is measured relative to acontrol sample, which control sample includes the MGAT1 polypeptide, theacyl donor, and the acyl acceptor, and no test agent. Typically, thereaction mixture includes magnesium ions (e.g., MgCl₂); and a buffer.Exemplary reaction conditions are provided in Example 5. Suitable acyldonors are fatty acyl CoA compounds and include, but are not limited to,oleoyl CoA. Typically, the acyl group of the acyl donor is labeled witha detectable label, such that when the acyl group is transferred to theacyl acceptor, the detectable label is also transferred, therebyallowing detection of the acylated acceptor molecule. Suitable acylacceptors are monoacylglyerols and diacylglycerols.

The DGAT2α or MGAT1 polypeptide in the screening assay may be purified,but need not be. For example, membrane fractions containing DGAT2α orMGAT1 polypeptides can be used.

Depending on the particular method, one or more of, usually one of, thecomponents of the screening assay may be labeled, where by labeled ismeant that the components comprise a detectable moiety, e.g. afluorescent or radioactive tag, or a member of a signal producingsystem, e.g. biotin for binding to an enzyme-streptavidin conjugate inwhich the enzyme is capable of converting a substrate to a chromogenicproduct. Where in vitro assays are employed, the various components ofthe in vitro assay, e.g. the substrate, the donor, the DGAT2α or MGAT1protein and the candidate inhibitor, etc. are combined in a assaymixture under conditions sufficient for DGAT2α or MGAT1 activity tooccur, as described in the experimental section, infra.

A variety of other reagents may be included in the screening assay andreaction mixture. These include reagents like salts, neutral proteins,e.g. albumin, detergents, etc that are used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used.

A variety of different candidate agents may be screened by the abovemethods. Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Using the above screening methods, a variety of different therapeuticagents may be identified. Such agents may target the enzyme itself, oran expression regulator factor thereof. Such agents may inhibitors orpromoters of DGAT2α or MGAT1 activity, where inhibitors are those agentsthat result in at least a reduction of DGAT2α or MGAT1 activity ascompared to a control and enhancers result in at least an increase inDGAT2α or MGAT1 activity as compared to a control. Such agents may befind use in a variety of therapeutic applications, as described ingreater detail below.

Methods and Compositions Having Medical Applications

The methods and compositions of the subject invention also have broadranging applications in a variety of medical applications, includingdiagnostic screening, therapeutic treatments of pathological conditions,in the regulation of DGAT2α or MGAT1 activity in desirable ways, and thelike.

The subject invention provides methods of screening individuals for apredisposition to a disease state or the presence of disease state,where such screening may focus on the presence of one or more markers,such as a mutated DGAT2α or MGAT1 gene or expression regulatory elementthereof, observed levels of DGAT2α or MGAT1; the expression level of theDGAT2α or MGAT1 gene in a biological sample of interest; and the like.

Samples, as used herein, include biological fluids such as blood,cerebrospinal fluid, tears, saliva, lymph, semen, dialysis fluid and thelike; organ or tissue culture derived fluids; and fluids extracted fromphysiological tissues. Also included in the term are derivatives andfractions of such fluids. The cells may be dissociated, in the case ofsolid tissues, or tissue sections may be analyzed. Alternatively alysate of the cells may be prepared.

A number of methods are available for determining the expression levelof a gene or protein in a particular sample. Diagnosis may be performedby a number of methods to determine the absence or presence or alteredamounts of normal or abnormal DGAT2α or MGAT1 in a patient sample. Forexample, detection may utilize staining of cells or histologicalsections with labeled antibodies, performed in accordance withconventional methods. Cells are permeabilized to stain cytoplasmicmolecules. The antibodies of interest are added to the cell sample, andincubated for a period of time sufficient to allow binding to theepitope, usually at least about 10 minutes. The antibody may be labeledwith radioisotopes, enzymes, fluorescers, chemiluminescers, or otherlabels for direct detection. Alternatively, a second stage antibody orreagent is used to amplify the signal. Such reagents are well known inthe art. For example, the primary antibody may be conjugated to biotin,with horseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to aflourescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

Alternatively, one may focus on the expression of DGAT2α- orMGAT1-encoding nucleic acids. Biochemical studies may be performed todetermine whether a sequence polymorphism in a DGAT2α or MGAT1 codingregion or control regions is associated with disease. Disease associatedpolymorphisms may include deletion or truncation of the gene, mutationsthat alter expression level, that affect the activity of the protein,etc.

Changes in the promoter or enhancer sequence that may affect expressionlevels of DGAT2α or MGAT1 can be compared to expression levels of thenormal allele by various methods known in the art. Methods fordetermining promoter or enhancer strength include quantitation of theexpressed natural protein; insertion of the variant control element intoa vector with a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence, e.g. a disease associated polymorphism.Where large amounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. Cells that express DGAT2αor MGAT1 may be used as a source of mRNA, which may be assayed directlyor reverse transcribed into cDNA for analysis. The nucleic acid may beamplified by conventional techniques, such as the polymerase chainreaction (PCR), to provide sufficient amounts for analysis. The use ofthe polymerase chain reaction is described in Saiki, et al. (1985),Science 239:487, and a review of techniques may be found in Sambrook, etal. Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.14.2–14.33. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. (1990), Nucl. Acids Res. 18:2887–2890; andDelahunty et al. (1996), Am. J. Hum. Genet. 58:1239–1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzedby one of a number of methods known in the art. The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to a wild-type DGAT2α or MGAT1 sequence. Hybridization with thevariant sequence may also be used to determine its presence, by Southernblots, dot blots, etc. The hybridization pattern of a control andvariant sequence to an array of oligonucleotide probes immobilized on asolid support, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence ofvariant sequences. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis (DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease, the sampleis digested with that endonuclease, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel or capillary electrophoresis, particularly acrylamideor agarose gels.

Screening for mutations in DGAT2α or MGAT1 may be based on thefunctional or antigenic characteristics of the protein. Proteintruncation assays are useful in detecting deletions that may affect thebiological activity of the protein. Various immunoassays designed todetect polymorphisms in DGAT2α or MGAT1 proteins may be used inscreening. Where many diverse genetic mutations lead to a particulardisease phenotype, functional protein assays have proven to be effectivescreening tools. The activity of the encoded DGAT2α or MGAT1 protein maybe determined by comparison with the wild-type protein.

Diagnostic methods of the subject invention in which the level of DGAT2αor MGAT1 expression is of interest will typically involve comparison ofthe DGAT2α or MGAT1 nucleic acid abundance of a sample of interest withthat of a control value to determine any relative differences, where thedifference may be measured qualitatively and/or quantitatively, whichdifferences are then related to the presence or absence of an abnormalDGAT2α or MGAT1 expression pattern. A variety of different methods fordetermining the nucleic acid abundance in a sample are known to those ofskill in the art, where particular methods of interest include thosedescribed in: Pietu et al., Genome Res. (June 1996) 6: 492–503; Zhao etal., Gene (Apr. 24, 1995) 156: 207–213; Soares, Curr. Opin. Biotechnol.(October 1997) 8: 542–546; Raval, J. Pharmacol Toxicol Methods November1994) 32: 125–127; Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216:299–304; Stolz & Tuan, Mol. Biotechnol. (December 19960 6: 225–230; Honget al., Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem.(1984) 143: 298. Also of interest are the methods disclosed in WO97/27317, the disclosure of which is herein incorporated by reference.

The subject diagnostic or screening methods may be used to identify thepresence of, or predisposition to, disease conditions associated withacylglycerol metabolism, particularly those associated with DGAT2α orMGAT1 activity. Such disease conditions include: hyperlipidemia(including excess serum triglyceride levels), cardiovascular disease,obesity, diabetes, cancer, neurological disorders, immunologicaldisorders, and the like.

Also provided are methods of regulating, including enhancing andinhibiting, DGAT2α or MGAT1 activity in a host. A variety of situationsarise where modulation of DGAT2α or MGAT1 activity in a host is desired,where such conditions include disease conditions associated with DGAT2αor MGAT1 activity and non-disease conditions in which a modulation ofDGAT2α or MGAT1 activity is desired for a variety of different reasons,e.g. cosmetic weight control.

For the modulation of DGAT2α or MGAT1 activity in a host, an effectiveamount of active agent that modulates the activity, e.g. reduces theactivity, of DGAT2α or MGAT1 in vivo, is administered to the host. Theactive agent may be a variety of different compounds, including: thepolynucleotide compositions of the subject invention, the polypeptidecompositions of the subject invention, a naturally occurring orsynthetic small molecule compound, an antibody, fragment or derivativethereof, an antisense composition, and the like.

The nucleic acid compositions of the subject invention find use astherapeutic agents in situations where one wishes to enhance DGAT2α orMGAT1 activity in a host, e.g. in a mammalian host in which DGAT2α orMGAT1 activty is low resulting in a disease condition, etc. The DGAT2αor MGAT1 genes, gene fragments, or the encoded DGAT2α or MGAT1 proteinor protein fragments are useful in gene therapy to treat disordersassociated with DGAT2α or MGAT1 defects. Expression vectors may be usedto introduce the DGAT2α gene or encoding nucleic acid into a cell. Suchvectors generally have convenient restriction sites located near thepromoter sequence to provide for the insertion of nucleic acidsequences. Transcription cassettes may be prepared comprising atranscription initiation region, the target gene or fragment thereof,and a transcriptional termination region. The transcription cassettesmay be introduced into a variety of vectors, e.g. plasmid; retrovirus,e.g. lentivirus; adenovirus; and the like, where the vectors are able totransiently or stably be maintained in the cells, usually for a periodof at least about one day, more usually for a period of at least aboutseveral days to several weeks.

Naturally occurring or synthetic small molecule compounds of interestinclude numerous chemical classes, though typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 50 and less than about 2,500 daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof. Ofparticular interest are those agents identified by the screening assaysof the subject invention, as described above.

Also of interest as active agents are antibodies that modulate, e.g.reduce, if not inhibit, DGAT2α or MGAT1 activity in the host. Suitableantibodies are obtained by immunizing a host animal with peptidescomprising all or a portion of a DGAT2 or MGAT1 protein, such as theDGAT2α or MGAT1 polypeptide compositions of the subject invention.Suitable host animals include mouse, rat sheep, goat, hamster, rabbit,etc. The origin of the protein immunogen may be mouse, human, rat,monkey etc. The host animal will generally be a different species thanthe immunogen, e.g. human DGAT2 used to immunize mice, etc.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part ofDGAT2α or MGAT1, where these residues contain the post-translationmodifications, such as glycosylation, found on the native DGAT2α orMGAT1. Immunogens comprising the extracellular domain are produced in avariety of ways known in the art, e.g. expression of cloned genes usingconventional recombinant methods, isolation from HEC, etc.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with DGAT2α of MGAT1, where the DGAT2α or MGAT1protein will preferably be in substantially pure form, comprising lessthan about 1% contaminant. The immunogen may comprise complete DGAT2α orMGAT1, fragments or derivatives thereof. To increase the immune responseof the host animal, the DGAT2 or MGAT1 may be combined with an adjuvant,where suitable adjuvants include alum, dextran, sulfate, large polymericanions, oil & water emulsions, e.g. Freund's adjuvant, Freund's completeadjuvant, and the like. The DGAT2α or MGAT1 may also be conjugated tosynthetic carrier proteins or synthetic antigens. A variety of hosts maybe immunized to produce the polyclonal antibodies. Such hosts includerabbits, guinea pigs, rodents, e.g. mice, rats, sheep, goats, and thelike. The DGAT2α or MGAT1 is administered to the host, usuallyintradermally, with an initial dosage followed by one or more, usuallyat least two, additional booster dosages. Following immunization, theblood from the host will be collected, followed by separation of theserum from the blood cells. The Ig present in the resultant antiserummay be further fractionated using known methods, such as ammonium saltfractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using DGAT2α or MGAT1 bound to an insolublesupport, protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal (1994) J.B.C. 269:26267–73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

For in vivo use, particularly for injection into humans, it is desirableto decrease the antigenicity of the antibody. An immune response of arecipient against the blocking agent will potentially decrease theperiod of time that the therapy is effective. Methods of humanizingantibodies are known in the art. The humanized antibody may be theproduct of an animal having transgenic human immunoglobulin constantregion genes (see for example International Patent Applications WO90/10077 and WO 90/04036). Alternatively, the antibody of interest maybe engineered by recombinant DNA techniques to substitute the CH1, CH2,CH3, hinge domains, and/or the framework domain with the correspondinghuman sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91–3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG 1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

In yet other embodiments of the invention, the active agent is an agentthat modulates, and generally decreases or down regulates, theexpression of DGAT2α- or MGAT1-encoding nucleic acids in the host.Antisense molecules can be used to down-regulate expression of thesetarget nucleic acids in cells. The anti-sense reagent may be antisenseoligonucleotides (ODN), particularly synthetic ODN having chemicalmodifications from native nucleic acids, or nucleic acid constructs thatexpress such anti-sense molecules as RNA. The antisense sequence iscomplementary to the mRNA of the targeted gene, and inhibits expressionof the targeted gene products. Antisense molecules inhibit geneexpression through various mechanisms, e.g. by reducing the amount ofmRNA available for translation, through activation of RNAse H, or sterichindrance. One or a combination of antisense molecules may beadministered, where a combination may comprise multiple differentsequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840–844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra, and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995), Nucl. Acids Res. 23:4434–42). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43–56.

As mentioned above, an effective amount of the active agent isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result, where the desired result in thedesired modulation, e.g. enhancement, reduction, of DGAT2α activity.

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredeffect. Thus, the agent can be incorporated into a variety offormulations for therapeutic administration. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols.

As such, administration of the agents can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulations to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, e.g. antisense composition, it may be introduced into tissuesor host cells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992),Anal Biochem 205:365–368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152–154), where gold microprojectiles arecoated with the DGAT DNA, then bombarded into skin cells.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

The subject methods find use in the treatment of a variety of differentdisease conditions involving acylglycerol metabolism, and particularlyDGAT2α activity, including both insufficient or hypo-DGAT2α activity andhyper-DGAT2α activity. Representative diseases that may be treatedaccording to the subject methods include: hyperlipidemia (includingexcess serum triglyceride levels), cardiovascular disease, obesity,diabetes, cancer, neurological disorders, immunological disorders, skindisorders associated with sebaceous gland activity, e.g. acne, and thelike.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as serum triglyceride level,weight, total body fat content, etc. As such, treatment also includessituations where the pathological condition, or at least symptomsassociated therewith, are completely inhibited, e.g. prevented fromhappening, or stopped, e.g. terminated, such that the host no longersuffers from the pathological condition, or at least the symptoms thatcharacterize the pathological condition. For example, where the diseasecondition is marked by the presence of elevated lipid levels, treatmentincludes at least a reduction in the observed lipid levels, including arestoration of normal lipid levels. As another example, where thedisease is obesity, treatment results in at least a reduction in theoverall weight and/or total body fat content of the host.

The subject methods also find use in the modulation of DGAT2α or MGAT1activity in hosts not suffering from a disease condition but in whichthe modulation of DGAT2α or MGAT1 activity is nonetheless desired. Forexample, sperm production in males has been associated with diglycerideacyltransferase activity. As such, in males where at least reduced spermproduction is desired, the subject methods can be used to reduce thistarget activity in such males, e.g. by administering an agent thatreduces DGAT2α activity in such males, where such agents are describedabove. In other words, the subject methods provide a means of malecontraception. Alternatively, where increased sperm count in a givenmale is desired, e.g. in those conditions where the male has reducedfertility, the subject methods can be used to enhance this targetactivity in the male and thereby increase sperm count and fertility,e.g. by administering to the male host a DGAT2α enhancing agent, asdescribed above.

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans.

Kits with unit doses of the active agent, usually in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

Methods and Compositions for Producing Diglycerides, DiglycerideCompositions, Triglycerides, and Triglyceride Compositions

Also provided by the subject invention are methods for preparingdiglycerides, diglyceride compositions, triglycerides, and triglyceridecomprising compositions, as well as the compositions produced by thesemethods.

In preparing triglycerides with the subject invention, at least thedirect substrates of the desired triacylglyercol, e.g. diacylglyceroland fatty acyl CoA, will be combined in the presence of the polypeptideunder conditions sufficient for the acylation of the diacylglycerol tooccur. The synthesis may occur in an in vitro system, e.g. in a vesselin which the substrates or precursors thereof and the DGAT2α enzyme, aswell as any other requisite enzymes (e.g. as need to convert thesubstrate precursors to substrates), or an in vivo system, e.g. a hostcell that naturally comprises the substrates and into which a DGAT2αgene or nucleic acid according to the subject invention has beeninserted in a manner sufficient for expression of the gene and provisionof the DGAT2α enzyme, where the resultant triglyceride products may beseparated from the host cell using standard separation techniques.

In preparing diglycerides with the subject invention, at least thedirect substrates of the desired diacylglyercol, e.g. monoacylglyceroland fatty acyl CoA, will be combined in the presence of the polypeptideunder conditions sufficient for the acylation of the monoacylglycerol tooccur. The synthesis may occur in an in vitro system, e.g. in a vesselin which the substrates or precursors thereof and the MGAT1 enzyme, aswell as any other requisite enzymes (e.g. as need to convert thesubstrate precursors to substrates), or an in vivo system, e.g. a hostcell that naturally comprises the substrates and into which an MGAT1gene or nucleic acid according to the subject invention has beeninserted in a manner sufficient for expression of the gene and provisionof the MGAT1 enzyme, where the resultant diglyceride products may beseparated from the host cell using standard separation techniques.

Of interest for use in producing di- and triglyceride compositions aretransgenic plants/fungi that have been genetically manipulated using thenucleic acid compositions of the subject invention to produce di- and/ortriglycerides and/or compositions thereof in one or more desirable ways.Transgenic plants/fungi of the subject invention are those plants/fungithat at least: (a) produce more diglyceride, diglyceride composition,triglyceride or triglyceride composition than wild type, e.g. producemore oil, such as by producing seeds having a higher oil content, ascompared to wild-type; (b) produce di- or triglyceride compositions,e.g. oils, that are enriched for di- or triglycerides and/or enrichedfor one or more particular di- or triglycerides as compared to wildtype; and the like. Of interest are transgenic plants that producecommercially valuable triglyceride compositions or oils, such as canola,rapeseed, palm, corn, etc., containing various poly- andmono-unsaturated fatty acids, and the like. Of particular interest aretransgenic plants, such as canola, rapeseed, palm, oil, etc., which havebeen genetically modified to produce seeds having higher oil contentthan the content found in the corresponding wild type, where the oilcontent of the seeds produced by such plants is at least 10% higher,usually at least 20% higher, and in many embodiments at least 30% higherthan that found in the wild type, where in many embodiments seeds havingoil contents that are 50% higher, or even greater, as compared to seedsproduced by the corresponding wild-type plant, are produced. The seedsproduced by such DGAT2α transgenic plants can be used as sources of oilor as sources of additional DGAT2α transgenic plants. Such transgenicplants and seeds therefore find use in methods of producing oils. Insuch methods, DGAT2α transgenic plants engineered to produce seedshaving a higher oil content than the corresponding wild-type, e.g. seedsin which the DGAT2α gene is overexpressed, are grown, the seeds areharvested and then processed to recover the oil. The subject transgenicplants can also be used to produce novel oils characterized by thepresence of triglycerides in different amounts and/or ratios than thoseobserved in naturally occurring oils. The transgenic plants/fungidescribed above can be readily produced by those of skill in the artarmed with the nucleic acid compositions of the subject invention. Seethe discussion on how to prepare transgenic plants, supra.

The triglyceride compositions described above find use in a variety ofdifferent applications. For example, such compositions or oils find useas food stuffs, being used as ingredients, spreads, cooking materials,etc. Alternatively, such oils find use as industrial feedstocks for usein the production of chemicals, lubricants, surfactants and the like.

Also of interest are transgenic non-human animals suitable for use assources of food products and/or animal based industrial products. Suchtransgenic non-human animals, e.g. transgenic mice, rats, livestock,such as cows, pigs, horses, birds, etc, may be produced using methodsknown in the art and reviewed supra. Such transgenic non-human animalscan be used for sources of a variety of different food and industrialproducts in which the triglyceride content is specifically tailored in adesirable manner. For example, such transgenic animals that have beenmodified in a manner such that DGAT2α activity is reduced as compared tothe wild type can be used as sources of food products that are low intriglyceride content, e.g. low fat or lean meat products, low fat milk,low fat eggs, and the like.

The following examples are offered primarily for purposes ofillustration. It will be readily apparent to those skilled in the artthat the formulations, dosages, methods of administration, and otherparameters of this invention may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

EXPERIMENT Example 1 Existence of DGAT2α

A. Mice (DGAT1−/−) lacking DGAT, as described in WO 99/67268 are leanand resistant to diet-induced obesity, but are still capable ofsynthesizing triglycerides (TG) and have normal plasma TG levels.However, DGAT activity is virtually absent in membrane preparations fromDGAT1−/−tissues (Smith et al., Nat.Genet. 2000 (25), 87–90). Using pulseassays in living cells, we measured that the residual TG synthesisactivity in DGAT 1−/−Mouse Embryonic Fibroblasts (MEF) or adipocytes wasabout 40% of that in wild-type cells. The results are graphicallydepicted in FIGS. 1A and 1B. In FIG. 1A the membrane fraction isolatedfrom MEF or adipocytes of wild-type or DGAT1−/−mice was used as theenzyme source in DGAT assays in vitro. In FIG. 1B living cells werepulse-labeled with [¹⁴C]oleic acid for 24 hours and [¹⁴C] incorporationin the TG fraction was measured.

In further assays, increased DGAT activity was observed inDGAT1^(−/−)membranes assayed without magnesium; and DGAT activity wasobserved to vary with magnesium concentration in liver and adiposetissue.

The above findings indicate the existence of DGAT2α, a second enzymewith diglyceride acyltransferase activity.

II. Mammalian DGAT2α Sequences

-   A. The human DGAT2α nucleic acid and amino acid sequences were    identified using standard procedures, as described above. The human    DGAT2α cDNA has the sequence appearing as SEQ ID NO:01, infra, while    the protein encoded thereby has the sequence appearing as SEQ ID    NO:02, infra.-   B. The mouse DGAT2α nucleic acid and amino acid sequences were    identified using standard procedures, as described above. The mouse    DGAT2α cDNA has the sequence appearing as SEQ ID NO:03, infra, while    the protein encoded thereby has the sequence appearing as SEQ ID    NO:04, infra.

Example 2 Characterization of DGAT2α

A. Molecular Weight

The mouse DGAT2α cDNA was determined to encode a 43 kD predicted proteinbased on the amino acid sequence. The mouse DGAT2α cDNA was determinedto have no sequence homology to DGAT1, as described in Cases et al.,supra. The mouse DGAT2α amino acid sequence was determined to have 2putative N-linked glycosylation sites. The mouse DGAT2α amino acidsequence was determined to have 6 putative PKC phosphorylation sites. AHydrophobicity plot assessed by Kyte-Doolittle (K-D) analysis revealedthe existence of multiple putative transmembrane domains in the mouseDGAT2α amino acid sequence. FIG. 2 provides a graphical result of thisanalysis. As such, there are regions of higher hydrophobicity compatiblewith the existence of one or more transmembrane domain.

Example 3 Expression of DGAT2α in Insect Cells

Sf9 insect cells were infected with wild-type baculovirus, mouseFLAG-tagged DGAT2α or mouse FLAG-tagged DGAT1 (Cases et al., supra)recombinant baculoviruses, and the membrane fractions were assayed forDGAT activity. The results are graphically provided in FIG. 3A. In FIG.3A a time course of DGAT2α virus infection is provided. Insect cellmembranes were isolated at the indicated times after infection.Expression of the FLAG-tagged DGAT2α protein was detected byimmunoblotting with an anti-FLAG antibody (Inset). DGAT activity wasmeasured at low (5 mM) or high (100 mM) magnesium concentration, using[¹⁴C]oleoyl CoA and cold diacylglycerol. The experiment was repeatedthree times and a representative experiment is shown. FIG. 3B shows thatDGAT2α activity is dependent on the presence of the diacylglycerolsubstrate. Assays were performed at low magnesium concentration, using[¹⁴C]oleoyl CoA with or without exogenous cold diacylglycerol. When nodiacylglycerol is added, no significant DGAT activity can be detectedover background. Data represent the mean (±SD) of three experiments. Tocompare the DGAT activity of DGAT1 and DGAT2α, membranes expressingequal levels of DGAT1 or DGAT2α (as assessed by immunoblotting with ananti-FLAG antibody) were assayed at low magnesium concentration usingincreased amounts of cold oleoyl CoA in the presence of exogenousdiacylglycerol. The results are provided in FIG. 3C. Lipids wereextracted and separated by TLC and TG accumulation was visualized bycharring and quantified by densitometry.

Example 4 Analysis of DGAT2α mRNA Expression in Various Tissues and inAdipocyte Differentiation

The tissue distribution of human DGAT2α mRNA was analyzed. The resultsare provided in FIG. 4.

DGAT2α expression increases during 3T3-L1 adipocyte differentiation.Mouse 3T3-L1 adipocyte differentiation was induced and mRNA wereisolated at the indicated times shown in FIG. 5. Quantitation of DGAT2αmRNA levels in triplicate samples was performed by Phosphorimageranalysis and corrected for loading relative to actin expression. Theresults are shown in FIG. 5.

Summary of DGAT2α

-   -   mouse DGAT2α has no sequence homology to DGAT1    -   mouse DGAT2α diacylglycerol acyltransferase activity inhibited        by high magnesium concentrations;    -   human DGAT2α RNA expression in many tissues, highest levels        found in liver, adipose tissue, and mammary gland    -   mouse DGAT2α markedly increased mRNA expression during 3T3-L1        adipocyte differentiation.

Example 5 Characterization of MGAT1 Enzymatic Activity

The two major pathways for synthesizing diacylglycerol are shown in FIG.9. FIG. 9 depicts the roles of DGAT and monoacylglycerolacyltransferases (MGAT) in the synthesis of triacylglycerides. Theexamples above describe the activity of DGAT2α. In the followingsection, the activity of MGAT1 is described.

MATERIALS AND METHODS

Cloning of MGAT1 cDNA. Mouse expressed sequence tags (ESTs) encodingMGAT1 were identified by BLAST database searches through their sequencehomology to DGAT2 from Mortierella rammaniana (accession no. AF391089).Based on these ESTs, primers were designed to amplify the completecoding sequence of MGAT1 from mouse liver RNA by reverse transcription(SuperScript Choice System, Gibco BRL, Rockville, Md.) and PCR (TakaraEx Taq, Panvera, Madison, Wis.). The MGAT1 sequence has been depositedin GenBank (accession no. AF384162).

Insect Cell Expression Studies. MGAT1 was tagged with an N-terminal FLAGepitope (MGDYKDDDDG, epitope underlined; SEQ ID NO:17) and expressed inSpodoptera frugiperda Sf9 insect cells as described. MGAT1 without FLAGwas also expressed to determine whether the presence of FLAG, whichpermits the detection and assessment of expression levels, alters MGAT1activity. Briefly, the MGAT1 coding sequence (with or without FLAG) wassubcloned into pVL1393 baculovirus transfer vector (PharMingen, SanDiego, Calif.). Recombinant baculoviruses were generated bycotransfecting Sf9 insect cells with the transfer vector and BaculoGoldDNA (PharMingen). High-titer viruses used for MGAT1 expression wereobtained after two rounds of amplification. FLAG-tagged-DGAT1 (accessionno. AF078752) and -DGAT2 (accession no. AF384160) were also expressed ascontrols. To prepare membrane fractions, cells were typically infectedwith virus for 3 days, washed with PBS, and homogenized by 10 passagesthrough a 27-gauge needle in 1 mM EDTA, 200 mM sucrose, 100 mM Tris-HCl,pII 7.4. Total membrane fractions (100,000×g pellet) were resuspended inhomogenization buffer and frozen at−80° C. until use. Expression ofMGAT1, DGAT2, and FLAG-tagged proteins in 5 μg of membrane protein wasverified by immunoblotting with an antiserum raised against theC-terminus (amino acids 295–316) of MGAT1, an antiserum against DGAT2,and an anti-Flag M2 antibody (Sigma, St. Louis, Mo.), respectively.

In Vitro Acyltransferase Assays. Generally, acyltransferase activitieswere assayed under apparent V_(max) conditions for 5 min in a finalvolume of 200 μl. Each reaction contained 100 μg of membrane proteins, 5mM MgCl₂, 1.25 mg/ml bovine serum albumin (BSA), 200 mM sucrose, 100 mMTris-HCl, pH 7.4, 25 μM acyl donor, and 200 μM acyl acceptor. Non-polaracyl acceptors (diacylglycerol, monoacylglycerol, cholesterol,phosphatidic acid, and sphingosine) were dispersed asphosphatidylcholine liposomes (molar ratio≈0.2), and polar acylacceptors (glycerol-3-phosphate, dihydroxyacetone phosphate,lysophosphatidic acid, and lysophosphatidylcholine) were dissolved inwater. Reactions were started by adding protein and terminated by adding4 ml of chloroform:methanol (2:1 v/v). The extracted lipids were dried,separated by TLC with hexane:ethyl ether:acetic acid (80:20:1 v/v/v),visualized with iodine vapor, and identified with lipid standards. Forexperiments with radiolabeled substrates, TLC plates were exposed tox-ray film to assess the incorporation of radioactivity into lipidproducts.

Specifically, DGAT activity was measured as described. Cases et al.(1998) Proc. Natl. Acad. Sci. USA 95:13018–13023. MGAT activity wasdetermined by measuring the incorporation of the [¹⁴C]oleoyl moiety intodiacylglycerol with 25 μM [¹⁴C]oleoyl CoA (specific activity, ˜20,000dpm/nmol) and 200μM exogenously added sn-2 monooleoylglycerol. In someassays, [¹⁴C]sn-1 monooleoylglycerol (specific activity, 18 μCi/μmol,200 μM final concentration, American Radiolabeled Chemicals, St. Louis,Mo.) was used as a radiolabeled tracer to measure MGAT activity in thepresence of unlabeled oleoyl CoA (25 μM). The dependence of MGAT1activity on monoacylglycerol and fatty acyl CoA as substrates wasdetermined by assaying MGAT1 with various concentrations of oleoyl CoAor monooleoylglycerol in the presence of 400 μM monooleoylglycerol or 50μM oleoyl CoA, respectively. Diacylglycerol mass was quantified bydensitometry after the lipid products were separated by TLC andvisualized by immersing the plate in a solution of 10% cupric sulfateand 8% phosphoric acid and heating at 180° C. for 30 min. Stereoisomersof monoacylglycerol (sn-1-, sn-2-monooleoylglycerol, and3-monostearoylglycerol) were from Sigma. MGAT activity in tissues wasmeasured in particulate fractions prepared from pooled tissues of three15-week-old male mice.

Mammalian Cell Expression Studies. For mammalian cell expression,FLAG-tagged MGAT1 was subcloned into a pcDNA3 vector and transfectedinto COS-7 or CHO cells with Fugene 6 (Roche Diagnostics, Chicago,Ill.). FLAG-tagged-DGAT1, -DGAT2, and -ACAT2 (cholesterolacyltransferase, accession no. AF078751) were expressed as controls.Membrane fractions were prepared as described for insect cells.Expression of FLAG-tagged proteins (in 20 μg of membrane proteins) wasverified by immunoblotting with the anti-FLAG M2 antibody. Forimmunocytochemistry, cells were grown and transfected on glass coverslips. Two days after transfection, cells were fixed in acetone:methanol(1:1) for 2 min and incubated in PBS containing 3% BSA and 0.2% TritonX-100 for 1 h at room temperature. Samples were then incubatedsequentially with 4 μg/ml anti-FLAG antibody (Sigma) for 1 h and 10μg/ml FITC-conjugated goat anti-mouse IgG (CalBiochem, Pasadena, Calif.)for 30 min. Antibodies were diluted in PBS containing 3% BSA and 0.02%Triton X-100. MGAT activities in membranes of transfected cells wereassayed as described above.

MGAT1 Tissue Expression Pattern in Mice. To determine tissuedistribution of MGAT1 expression, a mouse multiple tissue blot (SeeGene,Seoul, Korea), a blot of total RNA from indicated tissues, and a poly-A⁺RNA blot (CLONTECH, Palo Alto, Calif.) were hybridized with ³²P-labeledprobes generated by random priming (Amersham) with MGAT1 cDNA as thetemplate.

Results

Identification of an MGAT1 Gene. Mouse MGAT1 cDNA was originallyidentified as a DGAT candidate (DC) gene through its homology to genesencoding DGAT2. Mouse MGAT1 is also referred to herein, and in Cases etal. ((2001) J. Biol. Chem. 276:38870–38876), as mouse DC2. The aminoacid sequence of mouse MGAT1 is set forth in SEQ ID NO:06; and thenucleotide sequence encoding mouse MGAT1 is set forth in SEQ ID NO:05.The open reading frame of the MGAT1 cDNA predicts a 335-amino acidprotein, which is 40% identical to mouse DGAT2, as shown in FIG. 10A,and has a predicted molecular mass of 38.8 kDa. Like DGAT2, MGAT1contains sequences similar to a domain of phosphate acyltransferases.MGAT1 also possesses two putative N-linked glycosylation sites and apotential tyrosine phosphorylation site. The hydrophobicity plot forMGAT1 is similar to that for DGAT2 and predicts at least onetransmembrane domain (amino acids 21–43) in the amino terminus, as shownin FIG. 10B. Sequences for the human MGAT1 homologue have been reportedas human DC2, a member of the DGAT2 gene family. The mouse MGAT1 gene islocated on chromosome 1 (accession no. AC079223) and its human homologueis on chromosome 2 (accession no. NT_(—)005126).Mouse MGAT1 expressed in Insect Cells. To examine the biochemicalactivity of MGAT1 protein, we expressed FLAG epitope-tagged andnon-tagged versions of the cDNA in insect cells. The non-FLAG-taggedversion migrated on SDS-PAGE with an apparent molecular mass of ˜33 kDa.As expected, the FLAG-tagged version migrated slightly more slowlybecause of the FLAG epitope. Because MGAT1 shares sequence homology withDGAT2, we first examined whether membranes expressing MGAT1 have DGATactivity. With either [¹⁴C]dioleoylglycerol or [¹⁴C]oleoyl CoA as aradiolabeled substrate, MGAT1-expressing membranes incorporated moreradioactivity into triacylglycerols than membranes expressing wild-typeviral proteins or heat-inactivated MGAT1 indicating that these membraneshave DGAT activity. However, this DGAT activity was significantly lessthan that in control membranes expressing DGAT2, even though MGAT1protein was expressed at a higher level.

Since the assays with [¹⁴C]oleoyl CoA radiolabel revealed that asignificant amount of [¹⁴C]oleoyl CoA was incorporated intodiacylglycerol in membranes expressing MGAT1, we suspected that MGAT1possesses MGAT activity. To test this possibility, membranes expressingMGAT1 were assayed with either [¹⁴C]monooleoylglycerol or [¹⁴C]oleoylCoA as the labeled substrate. In both cases, membranes expressing MGAT1catalyzed the incorporation of the label into diacylglycerol,establishing that the MGAT1 protein possesses MGAT activity. This MGATactivity was confirmed by its dependence on MGAT substrates; whenunlabeled MGAT substrate (monooleoylglycerol or oleoyl CoA) was providedover a range of concentrations while the other substrate was heldconstant, the mass of diacylglycerol synthesized was dependent on theconcentration of substrate, as shown in FIG. 11. Further, theacyltransferase activity of MGAT1 appeared to be specific formonoacylglycerol (and possibly diacylglycerol) as the acyl groupacceptor; no acyltransferase activity was found in MGAT1-expressingmembranes when glycerol-3-phosphate, dihydroxyacetone phosphate,lysophosphatidate, lysophosphatidylcholine, sphingosine, or cholesterolwere used as the [¹⁴C]oleoyl CoA acceptor.

Next, since there are three stereoisomers of monoacylglycerol, wedetermined whether MGAT1 can acylate each of these stereoisomers. Inthese assays [¹⁴C]oleoyl CoA was used as the acyl donor and eithersn-1-monooleoylglycerol, sn-2-monooleoylglycerol, orsn-3-monostearoylglycerol as the acyl acceptor. Whensn-1-monooleoylglycerol or sn-3-monostearoylglycerol was used, the majorproduct was sn-1,3-diacylglycerol. On the other hand, whensn-2-monooleoylglycerol was used, the major product wassn-1,2(2,3)-diacylglycerol. These results indicate that MGAT1 canacylate each of the stereoisomers of monoacylglycerol, mainly at thesn-1 or sn-3 position. We also found that the specific activities ofMGAT1 using either sn-1-monooleoylglycerol or sn-2-monooleoylglycerol assubstrates were similar and that both increased proportionally withMGAT1 protein levels, as shown in FIG. 12. These findings indicate thatMGAT1 expressed in insect cells can use sn-1-monooleoylglycerol andsn-2-monooleoylglycerol equally well as substrates in vitro.

Mouse MGAT1 expressed in Mammalian Cells. To examine whether MGAT1protein expressed in mammalian cells also has MGAT activity, weexpressed mouse MGAT1 cDNA and control cDNAs transiently in monkeykidney COS-7 cells, as shown in FIG. 13. In cells expressing MGAT1,immunofluorescence microscopy demonstrated a perinuclear and reticularstaining pattern, consistent with distribution of the protein in theendoplasmic reticulum. COS-7 cell membranes expressing MGAT1incorporated more radioactivity into diacylglycerol when [¹⁴C]oleoyl CoAand sn-2-monooleoylglycerol were provided as substrates, indicating thatthese membranes possess MGAT activity. Similar levels of MGAT activitywere found when sn-1-monooleoylglycerol was used as the acyl acceptor.In contrast, MGAT activity was not detected in control membranes, exceptin those expressing DGAT1, which appeared to possess a low level of MGATactivity. Similar results were found when these cDNAs were expressed inChinese hamster ovary (CHO) cells.Tissue Distribution of Mouse MGAT1 Expression. MGAT1 mRNA expression washighest in the stomach and kidney; lower levels of expression werepresent in white and brown adipose tissue, uterus, and liver. MGAT1 mRNAexpression was not detected in the small intestine. Because MGATactivity had not previously been reported in mice, we performed MGATassays on membranes from mouse tissues. As demonstrated for many otherspecies, the highest activity was found in the small intestine, as shownin FIG. 14. MGAT activity was also detected at significant levels in thestomach, kidney, adipose tissue, and liver, where MGAT1 is expressed.

It is apparent from the above results and discussion thatpolynucleotides encoding mammalian DGAT2α and MGAT1 enzymes, as well asnovel polypeptides encoded thereby, are provided. The subject inventionis important for both research and therapeutic applications. Using theDGAT2α probes of the subject invention, the role of DGAT2α and itsregulation in a number of physiological processes can be studied invivo. The subject invention also provides for important new ways oftreating diseases associated with DGAT2α and MGAT, such ashypertriglycemia and obesity, as well as in the production oftryglycerides.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A polynucleotide present in other than its natural environmentencoding a polypeptide that exhibits monoacylglycerol and/ordiacylglycerol acyltransferase activity and comprising a nucleotidesequence that has at least 95% nucleotide sequence identity to thesequence set forth in SEQ ID NO:03.
 2. The polynucleotide according toclaim 1, wherein said encoded polypeptide exhibits diacylglycerolacyltransferase activity.
 3. An expression cassette comprising atranscriptional initiation region functional in an expression host, apolynucleotide according to claim 1 under the transcriptional regulationof said transcriptional initiation region, and a transcriptionaltermination region functional in said expression host.
 4. An isolatedcell comprising an expression cassette according to claim 3 as part ofan extrachromosomal element or integrated into the genome of a host cellas a result of introduction of said expression cassette into said hostcell.
 5. A method of producing a polypeptide that exhibitsmonoacylglycerol and/or diacylglycerol acyltransferase activity, saidmethod comprising: growing a cell according to claim 4, whereby saidpolypeptide is expressed; and isolating said polypeptide substantiallyfree of other proteins.
 6. The polynucleotide of claim 1, wherein saidencoded polypeptide exhibits monoacylglycerol acyltransferase activityand diacylglycerol acyltransferase activity.
 7. The polynucleotide ofclaim 1, wherein said encoded polypeptide has a length of from about 300amino acids to about 500 amino acids.
 8. The polynucleotide of claim 1,wherein said encoded polypeptide has at least about 98% amino acidsequence identity to SEQ ID NO:04.